Relationship of cytosolic ion fluxes and protein kinase C activation to platelet-derived growth factor induced competence and growth in BALB/c-3T3 cells

Platelet-derived growth factor (PDGF) and other agents that activate protein kinase C (PKC) rapidly alter cytosolic pH (pHi) and intracellular free calcium ([Ca++]i) in BALB/c-3T3 fibroblasts. To define whether changes in pHi or [Ca++]i are linked to PDGF-stimulated mitogenesis, these parameters were assessed in control and PKC depleted fibroblasts. PDGF addition to BALB/c-3T3 fibroblasts resulted in transient acidification of the cytoplasm followed by prolonged cytosolic alkalinization. Exposure of cells to 12-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that activates PKC, resulted in cytosolic alkalinization without prior acidification. Overnight incubation with 600 nM TPA decreased the total cell PKC histone phosphorylating activity in BALB/c-3T3 fibroblasts by greater than 90%. In PKC-deficient fibroblasts, TPA, and PDGF-induced alkalinization was abolished. In addition, the transient drop in pHi seen initially in control cells treated with PDGF is sustained to the point where pHi is fully 0.6-0.7 pH units below control cell values for up to 30 minutes. PDGF increased [Ca++]i threefold; this transient rise in [Ca++]i was only minimally affected (less than 15%) by lowering of the extracellular calcium level with ethylene glycol bis(b-aminoethyl ether)0 N,N,N' tetraacetic acid (EGTA) or blocking calcium influx with CoCl2. In contrast, 8-(diethylamine)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an agent thought to inhibit calcium release from intracellular stores, substantially inhibited the rise in [Ca++]i caused by PDGF. TPA and 1-oleoyl-2-acetylglycerol (OAG) increased [Ca++]i but in contrast to PDGF this effect was blocked by pretreatment of cells with EGTA or CoCl2. In PKC-deficient fibroblasts, PDGF still increased [Ca++]i and stimulated DNA synthesis as effectively as in controls. TPA and OAG however, no longer increased [Ca++]i. The continued ability of PDGF to stimulate DNA synthesis in the face of sustained acidification and the absence of PKC activity suggests that cytosolic alkalinization and PKC activation are not essential for PDGF-induced competence in BALB/c-3T3 fibroblasts.     

Platelet-derived growth factor-induced alterations in vinculin and actin distribution in BALB/c-3T3 cells

Exposure of BALB/c-3T3 cells (clone A31) to platelet-derived growth factor (PDGF) results in a rapid time- and dose-dependent alteration in the distribution of vinculin and actin. PDGF treatment (6-50 ng/ml) causes vinculin to disappear from adhesion plaques (within 2.5 min after PDGF exposure) and is followed by an accumulation of vinculin in punctate spots in the perinuclear region of the cell. This alteration in vinculin distribution is followed by a disruption of actin-containing stress fibers (within 5 to 10 min after PDGF exposure). Vinculin reappears in adhesion plaques by 60 min after PDGF addition while stress fiber staining is nondetectable at this time. PDGF treatment had no effect on talin, vimentin, or microtubule distribution in BALB/c-3T3 cells; in addition, exposure of cells to 5% platelet-poor plasma (PPP), 0.1% PPP, 30 ng/ml epidermal growth factor (EGF), 30 ng/ml somatomedin C, or 10 microM insulin also had no effect on vinculin or actin distribution. Other competence-inducing factors (fibroblast growth factor, calcium phosphate, and choleragen) and tumor growth factor produced similar alterations in vinculin and actin distribution as did PDGF, though not to the same extent. PDGF treatment of cells for 60 min followed by exposure to EGF (0.1-30 ng/ml for as long as 8 h after PDGF removal), or 5% PPP resulted in the nontransient disappearance of vinculin staining within 10 min after EGF or PPP additions; PDGF followed by 0.1% PPP or 10 microM insulin had no effect. Treatment of cells with low doses of PDGF (3.25 ng/ml), which did not affect vinculin or actin organization in cells, followed by EGF (10 ng/ml), resulted in the disappearance of vinculin staining in adhesion plaques, thus demonstrating the synergistic nature of PDGF and EGF. These data suggest that PDGF-induced competence and stimulation of cell growth in quiescent fibroblasts are associated with specific rapid alterations in the cellular organization of vinculin and actin.     

Platelet-derived growth factor-induced alterations in vinculin distribution in porcine vascular smooth muscle cells

Exposure of porcine vascular smooth muscle cells to platelet-derived growth factor (PDGF; 18-180 ng/ml) but not epidermal growth factor (EGF; 30 ng/ml), somatomedin C (SmC; 30 ng/ml), or insulin (10 microM), results in a rapid, reversible, time- and concentration-dependent disappearance of vinculin staining in adhesion plaques; actin-containing stress fibers also become disrupted following exposure of cells to PDGF. Disappearance of vinculin staining from adhesion plaques is also caused by 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 200-400 nM), though the time course of the disappearance of vinculin staining under these conditions takes longer than in cells exposed to PDGF. The PDGF-induced removal of vinculin from adhesion plaques was inhibited in a concentration-dependent fashion by 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8; 0.25-4 microM) and leupepetin (2-300 microM), and by n-alpha-tosyl-L-lysine chloromethylketone (TLCK; 100 microM) and trifluoperazine (TFP; 2.5 microM). Addition of PDGF to vascular smooth muscle cells caused a rapid, transient increase in cytosolic free calcium, from a basal resting level of 146 +/- 6.9 nM (SEM, n = 62) to 414 +/- 34 nM (SEM, n = 22) as determined using the calcium-sensitive indicator Fura-2 and Digitized Video Microscopy. This increase in cellular calcium preceded the disappearance of vinculin from adhesion plaques and was partially blocked by pretreatment of cells with TMB-8 but not leupeptin. This rise in cytosolic free calcium was found to occur in approximately 80% of the sample population and displayed both spatial and temporal subcellular heterogeneity. Exposure of cells to TPA (100 nM) did not result in a change in cytosolic free calcium. Both PDGF (20 ng/ml) and TPA (100 nM) caused cytosolic alkalinization which occurred after PDGF-induced disruption of vinculin from adhesion plaques, as determined using the pH-sensitive indicator BCECF and Digitized Video Microscopy. PDGF stimulated DNA synthesis and vinculin disruption in a similar dose-dependent fashion. Both could be inhibited by leupeptin or TMB-8. These results suggest that 1) exposure of vascular smooth muscle cells to PDGF is associated with the disruption of vinculin from adhesion plaques, 2) PDGF-induced vinculin disruption is regulated by an increase in cytosolic calcium (but not cytosolic alkalinization), and involves proteolysis; 3) activation of protein kinase C also causes vinculin removal from adhesion plaques but by a calcium-independent mechanism, and 4) the cellular response to PDGF-stimulated increases in cytosolic free calcium is heterogeneous.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interrelationships of platelet-derived growth factor isoform-induced changes in c-fos expression, intracellular free calcium, and mitogenesis

Both increases in c-fos proto-oncogene expression and intracellular free calcium ([Ca2+]i) have been implicated as necessary components of the signal transduction pathway by which platelet-derived growth factor (PDGF) stimulates DNA synthesis in cultured BALB/c3T3 fibroblasts. To determine the interrelationship between PDGF-induced increases in c-fos proto-oncogene expression and [Ca2+]i, purified, recombinant BB and AA homodimeric isoforms of PDGF were used to evaluate the dose-response relationships and mechanisms of growth factor-induced changes in these two parameters as well as DNA synthesis. Concentration-dependent increases in [Ca2+]i, c-fos expression, and [3H]thymidine incorporation were observed with both BB and AA PDGF isoforms. BB PDGF was consistently more potent and efficacious than the AA isoform in eliciting a given response. The [Ca2+]i dependency of PDGF-induced increases in c-fos expression and DNA synthesis was determined by pretreatment of cells with agents that inhibit increases in [Ca2+]i: BAPTA, Quin-2, and TMB-8. Under these conditions, PDGF-induced DNA synthesis was blocked, whereas c-fos expression was enhanced. Conversely, in cells made deficient in protein kinase C (PKC) activity by prolonged treatment with phorbol ester, BB and AA PDGF-induced c-fos expression was inhibited by 75-80%, while PDGF-induced increases in [Ca2+]i and DNA synthesis were unaffected or enhanced. Additionally, the PKC-independent component of PDGF-stimulated c-fos expression was found to be independent of increases in [Ca2+]i. These data suggest that 1) both BB and AA PDGF isoforms elicit alterations in [Ca2+]i and c-fos proto-oncogene expression through the same or similar mechanisms in BALB/c3T3 fibroblasts, 2) PDGF-stimulated increases in [Ca2+]i are not required for c-fos expression, and 3) distinct pathways regulate PDGF-induced c-fos expression and mitogenesis, with c-fos expression being substantially PKC-dependent yet [Ca2+]i-independent, while mitogenesis is [Ca2+]i-dependent yet PKC-independent.     

Identification of a BALB/c-3T3 cell protein modulated by platelet-derived growth factor.

The platelet-derived growth factor (PDGF) stimulates density-arrested BALB/c-3T3 cells to synthesize a protein (pII; Mr, 35,000) that is constitutively synthesized by spontaneously transformed BALB/c-3T3 (ST2-3T3) cells which do not require PDGF for growth. Antisera against a major excreted protein family (MEP) of retrovirus-transformed cells quantitatively precipitated cellular pII. PDGF-stimulated pII has the same molecular weight, a similar charge, and similar antigenic determinants as authentic MEP isolated from ST2-3T3 or retrovirus-transformed cells. MEP represented about 2% of the nonnuclear proteins synthesized by ST2-3T3 cells and 0.3 to 0.6% of the proteins synthesized by PDGF-treated BALB/c-3T3 cells, a three- to sixfold increase over the background. In BALB/c-3T3 cells, less PDGF was required for pII (MEP) synthesis than for DNA synthesis. PDGF induced a selective increase in pII (MEP) within 40 min. Such preferential synthesis was inhibited by brief treatment with actinomycin D, suggesting a requirement for newly formed RNA. The constitutive synthesis of pII (MEP) by ST2-3T3 cells was not inhibited by actinomycin D. Five spontaneously or chemical carcinogen-transformed tumorigenic BALB/c-3T3 cell lines were studied; they neither required PDGF for growth nor responded to it. These cell lines became arrested at confluence with a G1 DNA content. Each of these independently isolated lines synthesized pII (MEP) constitutively. Thus, the synthesis of pII (MEP) may be required, but is not sufficient, for PDGF-modulated DNA synthesis.     

Platelet-derived growth factor stimulation of [3H]-glucosamine incorporation in density-arrested BALB/c-3T3 cells

G0/G1 traverse in density-arrested BALB/c-3T3 cells is controlled by multiple serum-derived growth factors. Platelet-derived growth factor (PDGF) initiates a proliferative response, whereas factors present in plasma facilitate progression through G0/G1. In the absence of competence formation, progression factors are unable to stimulate cell cycle traverse. We have identified the stimulation of a biochemical process specific to competence formation in BALB/c-3T3 cells. PDGF treated BALB/c-3T3 cells incorporated 5-10-fold more [3H]-glucosamine (GlcN) into acid-insoluble material as compared to platelet-poor plasma (PPP) treated cultures. Increased GlcN incorporation occurred in density-arrested BALB/c-3T3 cells in response to treatment with other competence factors, fibroblast growth factor, and Ca3 (PO4)2 and was not due to cell-cycle traverse. Stimulation of [3H]-GlcN incorporation by PDGF was time dependent, and increased incorporation of [3H]-GlcN into protein required de novo protein synthesis. Several mechanisms through which PDGF could increase GlcN incorporation into cellular material were examined. Results of these studies suggest an increase in the cellular capacity to glycosylate proteins is a response to or a part of competence formation.     

Persistence of the mitogenic response to platelet-derived growth factor (competence) does not reflect a long-term interaction between the growth factor and the target cell

Quiescent BALB/c-3T3 cells exposed briefly to platelet-derived growth factor (PDGF) become "competent" to replicate their DNA even if PDGF is removed from cell culture medium prior to the onset of DNA synthesis. We have suggested that persistence of the PDGF-induced competent state reflects a rapidly induced and relatively stable biochemical change within the target cells. Others suggest that the phenomenon reflects a long-term association between PDGF and its target cells or perhaps between PDGF and the cell culture dish. This controversy has been addressed (a) by examining the effect of anti-PDGF antibodies on PDGF- induced competence and (b) by studying the chemical fate of 125I- labeled PDGF. Anti-PDGF antibodies inactive both soluble and surface- bound PDGF. However, if quiescent 3T3 cells are exposed to PDGF for as little as 30 min, subsequent addition of these antibodies to the culture medium does not prevent the mitogenic response. Under conditions where the PDGF-induced competent state decays stochastically with a t1/2 of 18-20 h, cell-associated 125I-PDGF decays with a t1/2 of approximately 50 min. These data do not support the concept that persistence of the PDGF-induced competent state reflects a long-term association between PDGF and the target cells or between PDGF and the culture dish.     

A transient rise in cytosolic calcium follows stimulation of quiescent cells with growth factors and is inhibitable with phorbol myristate acetate

We have used aequorin as an indicator for the intracellular free calcium ion concentration [( Ca++]i) of Swiss 3T3 fibroblasts. Estimated [Ca++]i of serum-deprived, subconfluent fibroblasts was 89 (+/-20) nM, almost twofold higher than that of subconfluent cells growing in serum, whose [Ca++]i was 50 (+/-19) nM. Serum, partially purified platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) stimulated DNA synthesis by the serum-deprived cells, whereas epidermal growth factor (EGF) did not. Serum immediately and transiently elevated the [Ca++]i of serum-deprived cells, which reached a maximal value of 5.3 microM at 18 s poststimulation but returned to near prestimulatory levels within 3 min. Moreover, no further changes in [Ca++]i were observed during 12 subsequent h of continuous recording. PDGF produced a peak rise in [Ca++]i to approximately 1.4 microM at 115 s after stimulation, and FGF to approximately 1.2 microM at 135 s after stimulation. EGF caused no change in [Ca++]i. The primary source of calcium for these transients was intracellular, since the magnitude of the serum-induced rise in [Ca++]i was reduced by only 30% in the absence of exogenous calcium. Phorbol 12-myristate 13-acetate (PMA) had no effect on resting [Ca++]i. When, however, quiescent cells were treated for 30 min with 100 nM PMA, serum-induced rises in [Ca++]i were reduced by sevenfold. PMA did not inhibit growth factor-induced DNA synthesis and was by itself partially mitogenic. We suggest that if calcium is involved as a cytoplasmic signal for mitogenic activation of quiescent fibroblasts, its action is early, transient, and can be partially substituted for by PMA. Activated protein kinase C may regulate growth factor-induced increases in [Ca++]i.     

beta-Interferon alters the pattern of proteins secreted from quiescent and platelet-derived growth factor-treated BALB/c-3T3 cells

Mouse beta-interferon (at a concentration of 100 units/ml or higher) inhibited the platelet-derived growth factor (PDGF)-induced replication of quiescent BALB/c-3T3 cells. The interferon treatment did not inhibit, but slightly enhanced, the accumulation of the following three PDGF-induced RNAs: myc RNA, JE RNA, and KC RNA. The treatment with interferon changed the pattern of secreted proteins from quiescent cells and from cells treated with partially purified PDGF; it inhibited the accumulation of the PDGF-induced proteins (including proteins of 63 and 32 kDa) and it induced the accumulation of several other proteins (including proteins of 89, 31.5, 30, and 10.5 kDa) in both quiescent and also in PDGF-treated cells.     

Cyclic AMP agonists induce the phosphorylation of phospholipase C-tau and of a 76 kDa protein co-precipitated by anti-(phospholipase C-tau) monoclonal antibodies in BALB/c-3T3 cells. Relationship to inositol phosphate formation.

Previous studies have demonstrated enhanced phosphorylation of phospholipase C-tau (PLC-tau), a key regulatory enzyme in phosphoinositide metabolism, in cells treated with platelet-derived growth factor (PDGF) and epidermal growth factor, both of which act via specific receptor tyrosine kinases. Our studies on BALB/c-3T3 cells show that agents that promote cellular cyclic AMP accumulation also increase the phosphorylation, specifically the serine phosphorylation, of this enzyme. Increased phosphorylation of PLC-t (2-3-fold) was evident within 5-10 min of addition of isobutylmethylxanthine (IBMX) and either cholera toxin or forskolin to cells, and persisted for at least 3 h. Treatment of cells with cyclic AMP agonists also enhanced, with similar kinetics, the phosphorylation of a 76 kDa protein co-precipitated by anti-PLC-tau monoclonal antibodies. Brief exposure of cells to cholera toxin/IBMX or forskolin/IBMX decreased inositol phosphate formation induced by the GTP-binding protein (G-protein) activator aluminium fluoride by approx. 50%, but was without effect on PDGF-stimulated inositol phosphate formation. These findings suggest that PLC-tau, and perhaps the 76 kDa co-precipitated protein, are substrates of cyclic AMP-dependent protein kinase in BALB/c-3T3 cells: however, the lack of effect of cyclic AMP elevation on PDGF-stimulated inositol phosphate formation indicates that the intrinsic activity of PLC-tau is unaltered by cyclic AMP-mediated phosphorylation.     

Post-transcriptional control of protein synthesis in Balb/c-3T3 cells by platelet-derived growth factor and platelet-poor plasma

Platelet-derived growth factor (PDGF) and platelet-poor plasma, which lacks PDGF, both induce a rapid increase in the rate of total protein synthesis within quiescent, density-arrested Balb/c-3T3 cells. This stimulation of protein synthesis is associated with an increased aggregation of ribosomes into polyribosomes. Nuclear functions are not required for this response, as demonstrated by the observation that this stimulation of protein synthesis occurs in cells pretreated with actinomycin D and in enucleated cells (cytoplasts). The response to PDGF persists even after PDGF has been removed from the culture medium, but in contrast, when plasma is removed from the medium, polysomes disaggregate and protein synthesis declines. PDGF and plasma do not function synergistically to increase protein synthesis, whereas they do to induce optimum DNA synthesis. Thus stimulation of the translational apparatus may be necessary for the mitogenic response of Balb/c-3T3 cells to growth factors, but it is not by itself sufficient.     

Dissociation of cellular transformation from platelet-derived growth factor independence

ST2-3T3, a spontaneously transformed BALB/c-3T3 cell line which does not require platelet-derived growth factor (PDGF) for growth, was fused to THO2, a PDGF-responsive non-transformed BALB/c-3T3 cell line, in order to learn whether transformation is expressed coordinately with PDGF independence. Hybrid cells were selected and grown in medium containing both HAT (hypoxanthine-aminopterin-thymidine) and ouabain; unfused cells of each parental type were killed in HAT-ouabain medium. Five independently isolated ST2-3T3xTHO2 hybrid cell lines were established and characterized for both transformation and PDGF responsiveness. All five were transformed, having a disorganized growth pattern and achieving a final cell density similar to that of ST2-3T3 cells. Two of these lines did not respond to a brief treatment with PDGF: the mitogen neither induced the synthesis of a PDGF-modulated lysosomal protein (termed MEP), nor stimulated the cells to enter the S phase; one line responded to PDGF by synthesizing both MEP and DNA, whereas two others synthesized MEP but not DNA. In contrast, four independently isolated cell lines obtained by fusing PDGF-responsive non-transformed BALB/c-3TC cells to the THO2 line were all PDGF-responsive for both MEP and DNA synthesis and were not transformed. It appears that PDGF independence is not required for the transformation of BALB/c-3T3 cells.     

Cell shape and increased free cytosolic calcium [Ca2+]i induced by growth factors

Growth factors stimulate DNA synthesis of neoplastic cells but not of non-neoplastic cells in suspension cultures. Similarly, growth ceases in dense monolayers of non-neoplastic cells, while crowded neoplastic cells continue to grow. The mechanism of these important phenotypic changes is unknown; the block in growth stimulation could occur in early events of signal transduction at the plasma membrane or in a late step in the final steps of gene activation and induction of DNA synthesis. One particular early intracellular event, [Ca2+]i increases, is in fact necessary for the induction of DNA synthesis in attached non-neoplastic Balb/c 3T3 cells stimulated by platelet-derived growth factor (PDGF). We therefore used digital image analysis of intracellular Fura-2 fluorescence to determine whether PDGF can stimulate [Ca2+]i transients in suspension or in dense monolayer cultures of Balb/c 3T3 cells. In dense cells (greater than 8 x 10(4) cells/cm2) the basal [Ca2+]i and [Ca2+]i response to PDGF stimulation were both lower than those in sparser, more spread cells. PDGF also did not release internal stores of Ca2+ or produce Ca2+ influx in completely suspended cells. Remarkably, attachment alone, with minimal cell spreading, was enough to reinitiate the entire early signalling mechanism stimulated by PDGF. Thus, a block in PDGF-induced [Ca2+]i increases may contribute to the inability of PDGF to stimulate DNA synthesis in suspended non-neoplastic cells. This early block in signal transduction must be abrogated in neoplastic cells growing in suspension and dense monolayer cultures.     

Phospholipase C gamma complexes with ligand-activated platelet-derived growth factor receptors. An intermediate implicated in phospholipase activation.

Several steps implicated in platelet-derived growth factor (PDGF) receptor-coupled signaling are activated by PDGF exposure at 0-4 degrees C. These include receptor self-phosphorylation, physical association with and phosphorylation of phospholipase C gamma (PLC gamma). Reduced temperature blocks PDGF internalization, making it possible to dissociate bound PDGF after PLC gamma tyrosine phosphorylation. We addressed the functional consequences of PDGF dissociation from intact cell PDGF receptors. PDGF exposure at 0-4 degrees C for 15 min stimulated self-phosphorylation of a subpopulation of BALB/c 3T3 cell PDGF beta-type receptors (35%) and initiated subsequent inositol phosphate production. A small fraction of cellular PLC gamma (1-3%) coprecipitated with ligand-activated PDGF receptors; 3-5% of cellular PLC gamma acquired phosphotyrosine. The PLC gamma coprecipitating with PDGF receptors did not contain detectible phosphotyrosine. Phosphotyrosine antibody recovered similar amounts of PLC gamma from soluble and particulate fractions of PDGF-stimulated cells. Acid dissociation of bound PDGF from receptor caused rapid dephosphorylation of PDGF receptors and PCL gamma, and interrupted PLC gamma-PDGF receptor coprecipitation. Orthovanadate blocked tyrosine dephosphorylation of both PDGF receptors and PLC gamma and stabilized coprecipitation. Orthovanadate reversed the acid wash effect to abrogate PDGF-stimulated inositol phosphate production. PDGF receptor remains competent to coprecipitate with PLC gamma and stimulate PLC-mediated inositol phosphate production if PDGF-induced receptor phosphorylation is maintained. Formation of a coprecipitable PDGF receptor-PLC gamma complex appears required for PDGF-stimulated inositol phosphate production.     

Control of cytolysis of BALB/c-3T3 cells by platelet-derived growth factor: a model system for analyzing cell death

In culture medium supplemented with 10% clotted blood serum, the saturation density of BALB/c-3T3 cells is determined jointly by cell replication and cell loss. By prelabelling cellular DNA with ³H-thymidine and also by time lapse photography, we studied cell loss independently of replication. Cell loss was accelerated when BALB/c-3T3 cells were transferred from serum-supplemented medium, which contains the platelet derived growth factor (PDGF), to medium supplemented with platelet-poor plasma which lacks it. Loss occurred via the disintegration of cell attached to the surface of the tissue culture dish. Cytolysis of individual cells occurred rapidly; less than 15 minutes transpired between the first indication of a perturbance (by phase contrast microscopy) and fragmentation of the cell cytoplasm. Kinetic analysis was consistent with random cell death rather than a fixed lifetime. The percentage of cells undergoing cytolysis was governed by the cell density; at high densities, such as are present in confluent cultures, a higher percentage of cell loss was noted than at low density. Cell death was antagonized by partially purified or electrophoretically homogenous preparations of-PDGF. Pure PDGF stimulated cell survivial at ng/ml in a concentration dependent fashion. The process of cell replication was not necessary for survival because PDGF prevented cytolysis in the presence of methotrexate, an inhibitor of DNA synthesis. A brief (4 hour) treatment with PDGF prevented cell death; such PDGF treated cells displayed increased survival after being taken up with trypsin and planted onto a fresh surface in plasma supplemented medium. Pituitary fibroblast growth factor, a functional analogue of PDGF for induc of DNA synthesis in BALB/c-3T3 cells, also functioned as an anticytolytic agent. By contrast, epidermal growth factor and insulin did not. Cytolysis of SV40-transformed cells occurred at a constitutively low rate and was insensitive to PDGF.     

Epidermal growth factor stimulates formation of inositol phosphates in BALB/c/3T3 cells pretreated with cholera toxin and isobutylmethylxanthine

Epidermal growth factor (EGF) stimulated the formation of inositol trisphosphate, inositol bisphosphate, and inositol phosphate in density-arrested BALB/c/3T3 cells pretreated for 1.5-4 h with cholera toxin, a potent activator of adenyl cyclase, and isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor. Concomitant addition of cholera toxin, IBMX, and EGF to cells did not increase inositol phosphate levels, and pretreatment with both agents was more effective than pretreatment with either alone. Pre-exposure of cells to cholera toxin and IBMX also enhanced the increase in inositol phosphates occurring in response to platelet-derived growth factor (PDGF). Preincubation of cells with cholera toxin and IBMX in the presence of cycloheximide abolished the effects of these agents on EGF- and PDGF-stimulated inositol phosphate production as well as the lesser increase in inositol phosphate formation produced by cholera toxin and IBMX in the absence of hormone. Preincubation of cells with cycloheximide did not affect EGF binding or the ability of PDGF to stimulate inositol phosphate formation. Cycloheximide also precluded EGF-induced inositol phosphate production when presented to cells 3 h after addition of cholera toxin and IBMX. These findings show that, under the appropriate conditions, EGF is capable of stimulating inositol phosphate formation in a nontransformed cell line.     

Biochemical and functional discrimination of platelet-derived growth factor alpha and beta receptors in BALB/c-3T3 cells.

Three biologically active isoforms of platelet-derived growth factor (PDGF) exist: PDGF-AB, the predominant form in human platelets; PDGF-BB, the product of the c-sis protooncogene; and PDGF-AA. PDGF-BB and PDGF-AB interact with two distinct PDGF receptors (termed alpha and beta) of similar size, whereas PDGF-AA binds alpha receptors only. To dissect alpha and beta receptor-mediated signals, we compared the biological activities of PDGF-AA and PDGF-BB in density-arrested BALB/c-3T3 cells, which possess a 4:1 ratio of beta to alpha receptors, and assessed the contribution of alpha receptors to PDGF-BB- and PDGF-AB-induced responses. In addition, we describe a convenient method for resolving alpha and beta receptors on one-dimensional protein gels. This protocol involves treatment of cells with neuraminidase, a desialylating agent, and subsequent in vitro autophosphorylation of solubilized cells, and was used to monitor the presence or absence of alpha and beta receptors under various experimental conditions. Our data show that although higher concentrations were required, PDGF-AA stimulated DNA synthesis to the same extent as did PDGF-BB. Both isoforms induced inositol phosphate formation, epidermal growth factor transmodulation, and PDGF receptor autophosphorylation; PDGF-AA, however, was less effective than was PDGF-BB even at doses causing maximal mitogenesis. Pretreatment of cells with PDGF-AA for 30-60 min at 37 degrees C effectively down-regulated alpha receptors as verified by the absence of desialylated alpha receptor phosphorylation. Depletion of alpha receptors did not affect the capacity of PDGF-BB or PDGF-AB to activate the beta receptor tyrosine kinase, as assessed by tyrosine phosphorylation of an endogenous substrate, or stimulate the formation of inositol phosphates. We suggest that alpha and beta receptors independently mediate similar biological responses in BALB/c-3T3 cells, and that alpha receptors are not required for responses induced by PDGF-BB or PDGF-AB.     

Conditional transformation mediated via a pertussis toxin-sensitive receptor signalling pathway.

To determine whether a cloned receptor coupled to pertussis toxin (PTx)-sensitive G-proteins can induce cell proliferation and oncogenic transformation, as observed for receptors that elicit PTx-insensitive enhancement of phosphatidyl inositol (PI)-specific phospholipase-C (PLC) activity, nontransformed murine BALB/c-3T3 cells were transfected with the rat serotonin-1A (5-HT1A) receptor. The 5-HT1A receptor is coupled to PTx-sensitive G-proteins to induce a cell-specific activation of PLC. While 1 microM 5-HT induced no change in PI turnover or cytosolic free calcium levels ([Ca2+]i) in receptor-negative nontransfected 3T3 cells, 5-HT induced a 2-fold increase in inositol trisphosphate accumulation and a 2.5-fold increase in [Ca2+]i in the 3T3-ZD8 clone, which expressed 0.6 +/- 0.2 pmol/mg protein of specific 5-HT1A binding sites. The stimulatory actions of 5-HT on PI turnover and [Ca2+]i in 3T3ZD8 cells displayed the pharmacology of the 5-HT1A receptor and were abolished by pretreatment with PTx. Thus, BALB/c-3T3 fibroblast cells express the PLC-linked pathway of the 5-HT1A receptor. Overnight treatment with 5-HT (1 microM) enhanced incorporation of [3H]thymidine into DNA extracted from serum-starved 3T3ZD-8 cells, an action that was also blocked by pretreatment with pertussis toxin. Long term (1-2 weeks) exposure to 5-HT in the medium led to phenotypic transformation of the cells, including the formation of foci with 1 microM 5-HT. These actions of 5-HT were not observed in untransformed 3T3 cells. We conclude that the PTx-sensitive PLC-linked pathway of the 5-HT1A receptor expressed in nontransformed BALB/c-3T3 cells, in concert with other serum-derived factors, predisposes the cells to enhanced proliferation and transformation.